NO158004B - PROCEDURE FOR THE PREPARATION OF A PREPARATION WITH CYTOLYTIC ACTIVITY. - Google Patents

Description

The present invention relates to a method for producing a preparation with cytolytic activity against prokaryotic and eukaryotic cells.

Ample evidence has shown that the combination

of Certain peroxidases with hydrogen peroxide and a halide ion provide a system with strong cytotoxic properties. The myeloperoxidase-hydrogen peroxide chloride system forms a powerful cytotoxic system that is effective against bacteria, fungi, viruses, mycoplasma and various mammalian cells. Similarly, the lactoperoxidase-hydrogen peroxide-thiocyanate system and the horseradish peroxidase-hydrogen peroxide-chloride system have been shown to have potent cytotoxic activity.

An equally cytotoxic system is obtained when a hydrogen peroxide-developing system is used instead of hydrogen peroxide. Thus, the glucose oxidase-horseradish peroxidase-chloride combination provides a powerful cytotoxic system when glucose is added. Galactose oxidase and xanthine oxidase have also been shown to be effective in this regard. Furthermore, it has been shown that the endogenous NADH oxidase activity of horseradish peroxidase is also able to activate the cytotoxic activity of the enzyme in the presence of chloride ions.

A large body of evidence indicates that cytotoxic systems such as those described above can be operative in polymorphonuclear leukocytes, eosinophils, macrophages and other cell types with cytotoxic properties. Such cells generally appear to use an NADH or NADPH oxidase as the peroxide-evolving enzyme.

Macrophages are a necessary component in the enhancement of natural cell-killing activity of Bacillus Calmette-Guerin (BCG) in mice. BCG also increases peroxide and superoxide production in macrophages. There is thus a possibility that the peroxidase system of the macrophages plays a role in enhancing the natural cell killer activity. Similarly, peripheral lymphocytes, which are predominantly T cells, contain a cytotoxic peroxidase. Chemiluminescence resulting from peroxide-evolving oxidative metabolism is observed when T lymphocytes are stimulated by Concanavalin A. Furthermore, immunization of mice with either soluble or particulate antigens causes an increase in peroxidase activity in the spleen that precedes the development of specific antibody . These observations suggest that oxidase and/or peroxidase activity on

somehow involved in the development of specific immune responses.

To date, none of the cytotoxic systems described above have been used in any in vivo experiments. However, some relevant attempts have been made some time ago by Schultz et al. Schultz, Snyder, Wer, Berger and Bonner; Chemical Nature and Biological Activity of Myeloperoxidase: Molecular Basis of Electron Transport, Academic Press, New York, N.Y., pp. 301-321 (1972); and Schultz, Baker and Tucker; Myeloperoxidase, Enzyme-Therapy of Rat Mammary Tumors; Cancer Enzymology, Academic Press, New York, N.Y. pp. 319-334 (1976).

Using mice bearing 20-methylcholanthrene-induced tumors, these authors injected myeloperoxidase in combination with thio-TEPA, an antitumor drug. They observed a significant reduction in tumor growth in the treated mice, but no complete regression. Neither myeloperoxidase nor thio-TEPA alone were effective in reducing tumor growth. The inhibition of tumor growth lasted as long as the treatment with myeloperoxidase and thio-TEPA was continued.

These results indicated that the activity of myeloperoxidase could play a role in the regulation of tumor growth, either directly or indirectly. Certain conclusions are difficult to draw on the basis of such experiments as the biological half-life of myeloperoxidase is only approx. 24 hours. It is worth noting that the toxic activity appeared to be specifically directed at the tumor tissue.

According to the invention, a method is provided for the production of a preparation which exhibits cytolytic activity against prokaryotic and eukaryotic cells, and which is characterized by a first oxidase enzyme and a second peroxidase enzyme which are capable of chemically cross-linking with the first enzyme and with a protein , for example bovine or human serum albumin, whereby both enzymes remain active, is dissolved in a buffer solution, after which a polymerizing agent is added and the solution polymerized,

and optionally an insoluble gel is frozen and lyophilized in isolation after cooling the polymerized solution, and optionally the gel is ground.

With the method according to the invention, a preparation comprising a first oxidase enzyme, a second peroxidase enzyme and a protein is produced, which first and second enzymes and which protein are chemically cross-linked, and which preparation exhibits cytolytic activity against prokaryotic and eukaryotic cells.

The prepared preparation is intended for use in cytolysis of prokaryotic and eukaryotic cells in living organisms. The preparation is thus produced as a mixture of a carrier medium and a composition exhibiting cytolytic activity towards prokaryotic and eukaryotic cells, which composition is formed by dissolving a first oxidase enzyme, a second peroxidase enzyme and a protein in a buffer solution, and polymerization of the composition by adding a polymerizing agent thereto. When used, the preparation is introduced into a living organism.

It is thus an aim of the invention to produce a preparation for use in a method for controlling tumor growth and infectious diseases in mammals.

It has been found that several peroxidases, when used in combination with a hydrogen peroxide and/or superoxide developing system, develop a strong cytotoxic activity when administered to tumor-bearing animals. It has further been found that this cytotoxic activity appears to be exclusively directed against neoplastic tissue.

In order to achieve the tumoricidal activity, it is necessary that the two enzymes are kept close to each other and that the enzymes are stabilized so that this significantly increases their half-life in vivo. Both of these requirements can be met by immobilizing the enzymes, either separately or in combination, on an insoluble carrier. This immobilization can be carried out by either chemically attaching the enzyme molecules to the insoluble support or by enclosing the enzyme molecules within it. molecular matrix of the carrier.

In this description, immobilization means the connections between active protein molecules and an insoluble macromolecule in any way that prevents the protein molecules from moving away from the insoluble carrier. An insoluble compound is defined as a compound that does not form a true solution in an aqueous medium at physiological pH values; in the aqueous media are aqueous buffer media as well as any body fluids. Compounds that form colloidal solutions in the above-mentioned aqueous media are considered to be insoluble compounds.

One or more injections of the above-described immobilized enzyme conjugate into tumor-bearing animals results in a partial or complete regression of the tumor tissue. It is advantageous that the material is injected into the tumor or close to the tumor, however, primary tumors as well as metastasized tumors are subjected to the cytotoxic effect of the immobilized enzymes.

It has further been found that the immobilized enzyme systems act as powerful activators of the specific immune response in tumor-bearing animals. This activation of the specific immune response may therefore be fully or partially responsible for the recurrence of the tumours. Similarly, the injection of potent inhibitors of the cytotoxic activity of the immobilized enzyme system results in a reduction of the specific immune response and makes the animals more susceptible to developing tumor growth.

The foregoing indicates that the immobilized enzyme system enhances and/or activates the natural mechanism that bodies use to eliminate transferred or foreign cells present in the body, while inhibitors of the cytotoxic system are able to inhibit this natural process.

response mechanism of the body against. the growth of abnormal cells.

For expedient reasons, the insoluble cross-linked cytotoxic oxidase-peroxidase system

hereinafter referred to as "ICCOPS". In addition, it should also be noted that three to four week old Sprague-Dawley rats were each injected intraperitoneally with 0.5 to 1.0 ml of Novikoff hepatobiliary suspension. The tumors were allowed to develop for five days or until ascites was established. At this point, individual animals were euthanized to determine the presence of tumor. The remaining tumor-bearing animals were then divided into test and control groups and treatment was started.

Preparation of ICCOPS

The cross-linked enzyme was prepared by dissolving 10 mg of horseradish peroxidase and 300 mg of bovine serum albumin or human serum albumin in a solution containing 2 ml of glucose oxidase (2400 units) and 2.5 ml of 0.1 M phosphate buffer, pH 6.9. The polymerization was initiated by adding 75 to 25% glutaraldehyde to the enzyme solution. The mixture was incubated for at least 24 hours at 4°C or until a gel was formed. The gel was immersed in 100 times its volume of distilled water. The water was decanted off and the gel was further washed with 100 additional volumes of distilled water over a sintered glass filter. The gel was then frozen and lyophilized. The lyophilized gel was ground in a pestle and mortar to a size that would pass through an 18 gauge hypodermic needle when swollen in phosphate buffered saline (PBS), pH 7.4.

The powdered ICCOPS was stored at -20°C until use.

Peroxidase activity of immobilized enzyme

Oxidase and peroxidase activity were qualitatively determined by adding 1 mg of the immobilized enzyme system to 0.1% glucose in PBS, pH 7.4, containing 1.5 mg ABTS per ml. If the green solution turned blue within an hour, the oxidase and peroxidase were considered active.

Procedures for the treatment of tumor-bearing rats

The main procedure for treatment with ICCOPS required

three consecutive treatment days with 5 mg of the preparation suspended in 1 ml of PBS, pH 7.4, containing 0.1% glucose. The suspension was injected into the abdominal cavity of rats which

bore 5-day-old tumors. Variations in the treatment systems were used as indicated in the results described later.

Test of cellular immunological response in rats

A single-cell suspension was prepared from spleen by passage through a stainless steel sieve. The cells were washed once in Hank's balanced salt solution (HBSS), and then suspended in a Tris-ammonium chloride solution. The Tris-ammonium chloride solution was prepared from 10 ml of 0.17 M Tris and 90 ml of 0.16 M ammonium chloride and the solution was adjusted to pH 7.2. The suspension was incubated in a water bath at 37°C to lyse the erythrocytes. The cells were centrifuged at 1000 rpm. minute in a "CRC 5000" DAMON/IEC Division centrifuge for 10 minutes and the supernatant was removed. The cells were washed in HBSS and again centrifuged. The pellet was suspended in modified Eagle's medium supplemented with 10% (V/V) normal rabbit serum, Hepes buffer 10 mM, non-essential amino acids (Gibco) and penicillin-streptomycin. A 100 µl aliquot of cells was removed and mixed with 100 µl 0.4% trypan blue. After three minutes, the cells and staining mixture were diluted with 19.8 ml of physiological saline resulting in a 1:200 dilution of cells. The cells were counted in a hemocytometer and their concentration adjusted to 2x10^ live cells/ml. Viability was greater than 90%.

The various antigens and mitogens were stored sterile at 4°C dissolved in PBS, pH 7.2, at concentrations of 1 mg/ml, 100 µg/ml and 10 µg/ml. Cultivations were carried out in standard 96-well microtiter plates constructed from tissue culture grade plastic. To the wells that required antigen to 1 µg/ml, 10 µl of antigen with a concentration of 10 µg/ml was added. To achieve final concentrations of 10 µg/ml or 100 µg/ml, for example, 10 µl of the concentration 100 µg/ml or 1 mg/ml was added. Other concentrations of different mitogens were prepared in a similar manner. The specific antigen used was "Keyhole Limpet Hemocyanin" (KLH) with which the rats were inoculated subcutaneously 14 days before the introduction of tumor cells. Each rat received 200 µg KLH in 0.1 ml PBS,

pH 7.6. Concanavalin A (ConA) was the mitogen used in the tests. The negative control consisted of the addition of 10

µl PBS at pH 7.2 to each control well. After addition of antigen or mitogen, each well received 100 µl of the cell suspension. The plates were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 24 hours. Then the wells were pulsed with 100 µl MEM containing tritiated thymidine at 5 µCi/ml, specific activity 2Ci/mmol. After 48 hours of cultivation, the cells were harvested on glass fiber filters using a "titertech" cell harvester. After drying, the filters were counted in a scintillation liquid consisting of toluene, PPO and

POP.

Obtained test results using the preferred embodiment dg process

Regression of Novikoff hepatomas in rats

Young female Sprague-Dawley rats were intraperitoneally inoculated each with 0.5 ml of a Novikof f hepatoma cell suspension. Five days later tumors had developed and in most cases the ascitic fluid was present in the peritoneal cavity.

At this point, each rat was injected intraperitoneally with 5 mg of ICCOPS suspended in 0.1% glucose solution. On the following day, the rats were again given 5 mg of ICCOPS, and this was repeated on the third day. Control experiments were carried out by injecting ICCOPS from which either the glucose oxidase or the peroxidase was omitted or by injecting only the glucose solution. Five days after the last treatment, four of the seven animals treated with the complete ICCOPS system were euthanized along with all the control animals that were still alive. All control animals had developed massive abdominal tumors amounting to as much as 25 g of tumor tissue. The animals treated with the complete ICCOPS system, on the other hand, showed the presence of some yellow fibrous connective tissue, but no evidence of active tumor growth. The three animals that were not euthanized remained alive for 8 months without signs of disease effects. (See Table I).

A total of 24 Novikoff hepatoma bearing rats were treated with the complete ICCOPS system. A complete regression of tumor tissue took place in each of the animals, and in all cases no disease effects appeared after the treatment.

Effect of ICCOPS, on hepatoma - tumors

A young female rat was inoculated intraperitoneally with 0.5 ml Niovikoff hepatoma cell suspension and the tumor was allowed to develop for 5 days. At this point the abdominal cavity was opened under anesthesia. Typical tumor development was observed accompanied by ascites. The abdominal cavity was then closed and 5 mg ICCOPS was injected intraperitoneally. The rat was then given two additional doses of 5 mg ICCOPS on the two subsequent days after the first administration, and the abdominal cavity was reopened on the tenth day after the last treatment. At this point, the tumor had transformed into a hard, nodular yellow fibrous mass. The rat reproduced two months later and produced a normal litter of young. This experiment illustrated that the peroxidase-activated tumor regression took place by means of tumor cell lysis. Most of the ICCOPS particles were found in the omental tissue. Apparently, there is little or no direct contact between the ICCOPS particles and the tumor cells, suggesting that the cytotoxic activity of the ICCOPS particles is transferred to the tumor cells via one or more soluble intermediates.

Histopathological analysis

A hematoxylin and eosin preparation of actively growing Novikoff hepatoma was examined. The tumor cells appeared to be of epithelial origin and little connective tissue stroma or encapsulation was present. The cells were poorly differentiated. The tumor consisted of a coherent whole of cells forming random small cystic-like structures. The tissue showed a random limited differentiation towards pseudorosettes of the cells around the vasculature, and in some areas a differentiation towards a papillary pattern. Mitotic figures were common. The vascularity was limited and there were numerous areas of necrosis present in the tissue. The tumor was therefore described as an undifferentiated carcinoma with certain characteristics of papillary cystic adenocarcinoma.

The same preparation of the yellow remains, of the tumor taken 10 days after the final treatment with ICCOPS was

investigated. The tissue consisted of islands of necrotic cells encapsulated by an active fibroblastic response. There was a marked breakdown of the neoplastic tissue and necrosis appears to be total. The tissue thus showed tissue necrosis with secondary fibroplasia.

The mesenteric lymph nodes in the same animal showed collapse due to an infiltration of neoplastic cells in the nodes.

The neoplastic cells had started the formation of isolated islands of tissue, which is an indication of active neoplastic metastasis. Thus, this animal had already undergone metastasis when the primary tumor had been destroyed. If this observation is representative of other animals, one can only conclude that any metastasized tumor was subsequently destroyed, as all remaining rats fully recovered.

Effect of peroxidase inhibitors

In order to obtain further evidence that the observed tumor regression was indeed activated by the ICCOPS system, an experiment was performed in which individual animals were injected with a potent peroxidase inhibitor. The DOPA analogue 3-aminotyrosine is a potent inhibitor of horseradish peroxidase with at 5 µM.

In this experiment, ICCOPS was administered to 8 hepatoma-bearing rats. 4 of the animals were also injected with 100 µg of 3-aminotyrosine.

The results indicated that the 3-aminotyrosine inhibited the action of ICCOPS and all four rats that received 3-aminotyrosine eventually died of the tumor, while the fourth rat that received only ICCOPS showed a complete remission. This confirms that the regression of hepatomas is indeed activated by

ICCOPS.

Effect of ICCOPS on normal tissue

Two of the male rats with regressed Novikoff hepatoma were sacrificed on the tenth day after the treatment with ICCOPS to assess the effects of the treatment on rapidly proliferating normal cells. For this purpose, the testicles of the animals were examined by electron microscopy and the ateroli cells were found and the spermatogenesis appears to be completely normal.

100 mg of ICCOPS was also injected into three normally healthy animals for three consecutive days. On the fourth day, one of the animals was killed. Microscopic examination showed that the ICCOPS particles were again embedded in the omental tissue. The other two rats were kept alive for a further six months. During this period, nothing was observed that could distinguish these from other normally healthy animals.

Effect of ICCOPS on other tumors

To determine whether or not the cytotoxic effect of ICCOPS in vivo is specific to rat adenocarcinomas, the effect of ICCOPS was tested on different types of tumors.

A malignant mouse melanoma (B16-F1) was grown in mice,

and after a significant amount of tumor cells were present in the animals, treatment with ICCOPS was started. The results showed that tumor necrosis occurred as a result of the ICCOPS treatment. However, treatment for four to five consecutive days was necessary to achieve complete remission.

Several spontaneous rat mammary tumors were also treated. In these cases, 5 mg of ICCOPS was injected near the tumor for three to four consecutive days. A biopsy taken ten days after the last day of treatment showed that complete remission of the tumor had taken place. Pathological examination determined that both a spontaneous breast carcinoma and a breast sarcoma could be successfully treated with ICCOPS.

Furthermore, a dog with advanced Hodgkin's disease (a malignant lymphoma) was treated. ICCOPS was injected directly into one of the diseased lymph nodes for three days. At this point the dog died. However, severe necrosis of the lymphoma was observed in the treated lymph nodes indicating that this lymphoma. is also sensitive to the impact of ICCOPS.

Similarly, a complete remission of a benign tumor (a fibroadenoma) in a rat was also achieved.

These data indicate that at least several types of tumors are affected by the cytotoxic activity of ICCOPS, and that animals bearing such tumors can be successfully treated in this way. However, such data indicate that not all tumors respond equally well to ICCOPS treatment; a different treatment regimen is necessary for each type of tumor to achieve complete remission.

Antitumor activity of other peroxidases

The use of lactoperoxidase instead of horseradish peroxidase in the ICCOPS preparation provides a product which is also effective in promoting tumor regression in vivo. Its in vivo activity against rat hepatomas is somewhat less than that of the horseradish peroxidase system; administration had to be continued for four consecutive days to achieve the same results as three days of ICCOPS administration.

These results show that the tumoricidal activity is not specific for horseradish peroxidase but can therefore be promoted by certain other mammalian peroxidases.

Enhancement of tumor growth by aminotyrosine

To assess whether or not endogenous peroxidases play a role in the natural resistance to a neoplastic growth, individual rats were injected with a strong inhibitor of the cytotoxic activity of peroxidases and the animals were then challenged with a suspension of hepatoma cells.

Four adult male rats were injected intraperitoneally with 1 mg of 3-aminotyrosine in 1 ml of water. Four control rats were injected with 1 ml of water. On the following day, all rats were injected with 1 ml of a diluted Novikoff hepatoma cell suspension; in which the number of cells was normally insufficient to induce tumor growth. On the same day, another 1 mg of aminotyrosine was administered to the rats that had received the inhibitor the previous day, and this was repeated for another three days. The four control rats similarly received water

injections.

On the ninth day after tumor inoculation, all rats were sacrificed and examined for the presence of tumor. No tumor was present in any of the control animals.

On the other hand, each of the animals treated with aminotyrosine developed tumor growth. One rat had a large central omental tumor, while the other three rats contained a number of small tumors scattered throughout the mecentral omentum and over the surface of the small intestine.

This dramatic difference indicates that 3-aminotyrosine inhibits the natural mechanism by which the animals protect themselves against neoplasia. As such, this action is similar to that of various immunosuppressive compounds.

Effects of ICCOPS on the immune system

Table II illustrates the change in response to Concanavalin A stimulation of spleen cells obtained from tumor-bearing rats and from rats with regressed tumors compared to normal healthy animals.

The data show that spleen cells from tumor-bearing animals

are very active in the incorporation of thymidine and are insensitive to further stimulation by Concanavalin A. Even at low concentrations the mitogen actually acts as an inhibitor. Spleen cells obtained from animals with relapsed tumors, on the other hand, can be stimulated by the mitogen, although they are more active than the cells from healthy rats.

When spleen cells from animals with actively regressing tumors were tested, a pattern similar to that of animals with fully regressed tumors was observed. These results suggest that the changes in the immune response occur rapidly after the administration of ICCOPS. Similar results were obtained with spleen cells from rats previously immunized with KLH. (Table III). Cells from animals with actively relapsing tumors again behaved more like cells from normal animals in their response to stimulation with KLH than cells from tumor-bearing animals.

These data indicate that changes in the immune system occur simultaneously with tumor recurrence as a result of the action of the peroxidase system. Whether these changes occur as a result of the relapsing tumor or are directly caused by the presence of the peroxidase system cannot be determined at present.

Conclusion

The data presented in the present description show that ICCOPS, an immobilized system with glucose oxidase and horseradish peroxidase as its functional components, rapidly and selectively destroys a highly malignant and advanced adenocarcinoma in rats. In addition, other malignant tumors, including melanoma, sarcoma, and lymphoma are subject to selective destruction by ICCOPS. Previous in vitro studies by others using soluble glucose oxidase and horseradish peroxidase showed no specificity towards tumor cells compared to normal cells. A similar lack of specificity was observed with the myeloperoxidase and lacto-peroxidase systems. In an in vivo study, on the other hand, a very high degree of selectivity was obtained as illustrated by the following two facts.

First, no obvious damage was found in normal tissue, including rapidly proliferating testicular cells; and secondly, histopathological tissue sections showed that fibroblasts located in close proximity to necrosing tumor cells are mainly normal.

It was also shown that peroxidases different from horseradish peroxidase can be used in ICCOPS. Tumor regression was obtained using lactoperoxidase and cytotoxic activity in vitro was demonstrated using myeloperoxidase and snakeskin peroxidase.

The function of the glucose oxidase in the ICCOPS system is only to provide the hydrogen peroxide and/or superoxide needed to convert the peroxidase to the cytotoxic form. Glucose oxidase was chosen for practical reasons as its substrate (glucose and oxygen) is readily available for its use in vivo. However, its function can be fulfilled by any system that produces either hydrogen peroxide or superoxide.

It was also found that the peroxidase system activates a secondary antitumor mechanism. This secondary system appears to be the cell-mediated part of the immune system. The spontaneous blastogenic activity of spleen cells from rats with actively growing tumors indicates a maximally stimulated immune system that fails to produce an effective response. Introduction of ICCOPS, however, rapidly modifies the immune system by reducing the ineffective response and restoring the mitogenic and specific immune response. These results indicate that ICCOPS acts as an immunostimulant in animals whose normal response is inadequate. The increased sensitivity of the animals to a challenge with tumor cells as a result of 3-aminotyrosine administration, which thus acts as an immunosuppressive agent, also supports this conclusion.

Thus, ICCOPS as well as peroxidase inhibitors can be useful as therapeutic agents in diseases that affect the immune system, in regulation of the immune system after organ transplants and other tissue implantations, as well as in every therapy that requires either a suppression or

activation of the endogenous immune response.

There are of course many variations on the specific steps, techniques, doses, materials and the like that can be carried out in the work described here.

Claims (4)

1. Method for producing a preparation with cytolytic activity against prokaryotic and eukaryotic cells, characterized in that a first oxidase enzyme and a second peroxidase enzyme capable of chemically cross-linking with the first enzyme and with a protein, for example bovine or human serum albumin, whereby both enzymes remain active, are dissolved in a buffer solution, after which a polymerizing agent is added and the solution is polymerised, and optionally an insoluble gel isolated after cooling of the polymerised solution is frozen and lyophilised, and optionally the gel is ground up. 2. Method according to claim 1, characterized in that a glucose oxidase is used as the first enzyme. 3. Method according to any one of the preceding claims, characterized in that horseradish peroxidase is used as the second enzyme. 4. Method according to any one of <i>claims 1 to 3, characterized in that lactoperoxidase is used as the second enzyme.